The RH family is composed of multiple polytopic membrane proteins present in a wide variety of species ranging from the most primitive unicellular slime mold to man. Two members, the variable RHCED polypeptides and the invariant RHAG glycoprotein, are restricted to red cells, defining the clinically important RH blood group system. These proteins have an important but unidentified function, as evidenced by changes in red cell morphology and physiology by products of their mutated genes. Based on our preliminary studies, we hypothesize that the RHAG homologs possess a conserved transport function in both erythroid and nonerythroid cells, while the RHCED series perform a related or modified function confined only to erythroid cells. We will examine this hypothesis by using model systems amenable to biochemical experimentation and genetic manipulation. Our long-term objectives are to define the molecular cellular requirement for RH expression, elucidate the function of the RH family proteins possibly as membrane transporters and unravel the individual and collective functional roles of RHAG/RHCED in red cells. To meet these goals, our specific aims are as follows. 1) To define the molecular cellular requirement for RH expression, we will use Rh-null mutations as a natural model and determine their mechanistic effects on cellular routing, membrane insertion and protein interaction. 2) To study the possible transport function, the slime mold ortholog RhgA will be employed as a simple model system. We will assess the function of RhgA by insertional gene inactivation and study the null mutants by expression rescue with wild type RhgA and human RH proteins. Candidate ligands will be sought and tested in transport assays. 3) To unravel the individual and collective functions of RHAG/RHCED, we will target-disrupt by homologous recombination the Rhced and Rhag orthologs in mice and determine the mutant red cell phenotypes. The proposed studies should lead to identification of the long-sought functional role of the RH family of proteins, and provide new insight into the structure/function relationships. The knowledge gained will also deepen our understanding of clinical problems associated with the RH blood group system, allowing the clinical service to explore new approaches for their management.